title: Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation.
abstract: The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.
authors.affiliation: Division of Biochemistry and CancerGenomiCs.nl, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands, European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany, Institute for Biochemistry, Justus Liebig University, Heinrich-Buff Ring 58, D-35392, Giessen, Germany, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA, Department of Cell Biology and Genetics, Cancer Genomics Center, Erasmus Medical Center, PO Box 2040, 3000 CA Rotterdam, the Netherlands and Department of Radiation Oncology, Erasmus Medical Center, PO Box 2040, 3000 CA Rotterdam, the Netherlands.
authors.forename: Flora S
authors.initials: FS
authors.lastname: Groothuizen
authors.forename: Alexander
authors.initials: A
authors.lastname: Fish
authors.forename: Maxim V
authors.initials: MV
authors.lastname: Petoukhov
authors.forename: Annet
authors.initials: A
authors.lastname: Reumer
authors.forename: Laura
authors.initials: L
authors.lastname: Manelyte
authors.forename: Herrie H K
authors.initials: HH
authors.lastname: Winterwerp
authors.forename: Martin G
authors.initials: MG
authors.lastname: Marinus
authors.forename: Joyce H G
authors.initials: JH
authors.lastname: Lebbink
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.forename: Peter
authors.initials: P
authors.lastname: Friedhoff
authors.forename: Titia K
authors.initials: TK
authors.lastname: Sixma
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli